TREASURY DEPARTMENT. 

Public Health and Marine-Hospital Service of the United States. 

Walter Wyman, Surgeon-General. 

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1 HYGIENIC LABORATORY-BULLETIN No. 11. 

N1. J. ROSENAU. Director. 

_ February, 1903. 

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AN EXPERIMENTAL INVESTIGATION 
OF TRYPANOSOMA LEWISI. 


BY 


EDWARD FRANCIS. 



WASHINGTON: 

GOVERNMENT PRINTING OFFICE. 
I9O3. 


Monograph 

























































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TREASURY DEPARTMENT. 

Public Health and Marine-Hospital Service of the United States. 

Walter Wyman, Surgeon-General. 


HYGIENIC LABORATORY-BULLETIN No. 11. 

XI. J. KOSENAU, Director. 

February, 1903. 


AN EXPERIMENTAL INVESTIGATION 
OF TRYPANOSOMA LEWISI. 


EDWARD FRANCIS. 



WASHINGTON: 

GOVERNMENT PRINTING ' OFFICE. 
I903. 

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ORGANIZATION OF HYGIENIC LABORATORY. 

Walter Wyman, Surgeon-General , 

U. S. Public Health and Marine-Hospital Service. 

ADVISORY BOARD. 

-, U. S. Army; Surg. John F. Urie, U. S. Navy; Dr. D. E. Salmon, 

Chief of U. S. Bureau of Animal Industry; and Milton J. Rosenau, U. S. 
Public Health and Marine-Hospital Service, ex officio. 

Prof. William H. Welch, Prof. Simon Flexner, Prof. Victor C. Vaughan, Prof. 
William T. Sedgwick, and Prof. Frank F. Wesbrook. 

LABORATORY CORPS. 

Director. —P. A. Surg. Milton J. Rosenau. 

Assistant Director. —P. A. Surg. John F. Anderson. 

Pharmacist. —M. H. Watters, Ph. G. 

DIVISION OF PATHOLOGY AtfD BACTERIOLOGY. 

Chief of Division. —P. A. Surg. Milton J. Rosenau. 

Assistants. —P. A. Surg. John F. Anderson and H. B. Parker, and Asst. Surg. 
Thomas B. McClintic, Clarence W. Wille, and Edward Francis. 

DIVISION OF ZOOLOGY. 

Chief of Division.— Ch. Wardell Stiles, Ph. D. 

Assistants. —Philip E. Garrison, A. B.: Brayton H. Ransom, B. Sc., A. M.; Earle 
C. Stevenson, B. Sc.; Arthur L. Murray. 

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CONTENTS 


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Page. 

1. Introduction.. 5 

2. Occurrence and morphology of Trypanosoma Lewisi _ 6 

3. Cycle of development_ 7 

4. Multiplication by— 

(a) Transverse division_ 8 

( b ) Longitudinal division_____ 8 

(c) Segmentation_ 8 

5. Duration of the infection__ 9 

6. Motion of the trypanosome __ 10 

7. Symptoms in rats_ 10 

8. Staining of the parasite_ 11 

9. Active immunity_ 13 

10. Passive immunity_ 13 

11. Susceptibility of animals to Trypanosoma Lewisi _ 14 

12. Autoagglutination_ 14 

13. Agglutination- 16 

(а) By immune serum_ 16 

(б) By normal sera ___ _ 17 

(c) In the ice chest..... 18 

14. Transmission of the disease by— 

1. Intraperitoneal inoculation .. 18 

2. Subcutaneous inoculation_ 19 

3. Intrastomachal injection_ 20 

4. Feeding.. 20 

5. Fleas. 21 

6. Lice. 21 

15. Trypanosomes in Stegomyiafasciata - 21 

16. The parasites outside the body_ 22 

17. The trypanosome of the hamster- 23 

18. Trypanosomiasis as a disease of man (11 cases)_ 24 


3 















































AN EXPERIMENTAL INVESTIGATION OF 
TRYPANOSOMA LEWISI. 


By Edward Francis, 

Assistant Surgeon , U. S. Public Health and Marine-Hospital Service. 


The great importance of a thorough familiarity with trypanosomes 
is seen when we consider that within the past year and a half the 
recognition of trypanosomes was forced upon our governmental medi¬ 
cal officers in the Philippines, where the army horses and mules were 
dying in great numbers, due to the presence in their blood of a para¬ 
site which the untrained eye might readily regard as a spirillum or a 
filaria, but which proved to be the trypanosome of Surra (1). It is 
probably best in the beginning to say something of trypanosomes in 
general. They are animal parasites (Hematozoa) of large size which 
are often found in enormous numbers free in the blood plasma, but 
do not invade the interior of the blood corpuscles. They manifest a 
strikingly vigorous eel-like motility as they dart among the corpuscles, 
pushing them aside. 

The Trypanosoma Evansi is the cause of surra, a fatal disease of 
horses and mules in India and the Philippines. 

The Trypanosoma Brucii causes the tsetse fly disease, or Nagana, 
which attacks the horses and cattle in central Africa. 

The Trypanosoma equinum is the cause of mal de caderas, a disease 
of horses, etc., in South America. 

Dourine, or maladie du Colt , a disease of horses and dogs, particu¬ 
larly in Algeria and Spain, has been attributed to the Trypanosoma 
equiperdum. 

The Trypanosoma Lewisi is the cause of a nonfatal affection of 
wild rats, which harbor spontaneously the parasites in enormous 
numbers in their blood plasma. 

And now within the past year we have a trypanosome of man. 

Whether the six trypanosomes mentioned above represent really 
distinct species or whether two or more of them will be found to be 
identical must remain for further scientific investigation. Morphol- 
logically all six have many structures in common, but they also show 

5 




6 


certain differences. Still, an accurate differential diagnosis on the 
basis of structure alone is not altogether practicable. 

As a harmless inhabitant of the blood still other trypanosomes are 
harbored by the hamster, certain fish, frogs, and birds. 

OCCURRENCE AND MORPHOLOGY OF TRYPANOSOMA LEWISI. 

Our studies were made entirely with the rat trypanosome ( Trypan¬ 
osoma Lewisi). The natural host is the wild gray rat and the sewer 
rat. These rats harbor the trypanosomes in their blood and they 
infect each other spontaneously. 

Crookshank (2) investigated a number of rats in London and found 
that 25 per cent of the apparently healthy rats harbored trypanosomes. 

Rabinowitsch and Kempner (3) examined the blood of fifty wild 
rats caught about the city of Berlin and found that eighteen of them 
were infected with trypanosomes. 

Laveran and Mesnil (4) found two cases of infection among forty- 
three sewer rats examined in Paris. 

We examined sixty of our home rats caught in different parts of 
Washington and found no cases of infection. 

Natural infection of the white or spotted rats has never yet been 
found, although they are very susceptible to the infection by inocula¬ 
tion. We inoculated the trypanosome into white rats and examined 
the blood in fresh and stained preparations. 

The parasite may be considered in three parts: The body, the 
undulating membrane, and the flagellum. The body is the elongated 
portion which has attached to its side the undulating membrane and 
to one of its ends the flagellum. The body of the adult parasite 
measures 13 to 25 micra in length, which is about two to three times 
the diameter of a red blood corpuscle. Its breadth is 2 to 4 micra. 
The body runs to a beak-like process at one end, while at the other 
end there is a free flagellum 7 to 15 micra in length. The flagellate 
end is considered the anterior or front end because locomotion takes 
place in the direction of the flagellum, while the beak end is desig¬ 
nated the hind end, since it follows the body in locomotion. In the 
hinder fourth of the body, about 4 micra from the end, there is a 
short rod-shaped structure, the long axis of which is at a right angle 
to the long axis of the body; this spot is called the centrosome. It 
can with difficulty be seen in a fresh preparation. In the anterior 
fourth of the body of the parasite there is a larger strongly retractile 
spot, which is regarded as the nucleus. 

The undulating membrane is a clear, fine, transparent membrane 
which presents an attached border, a free border, and a web. It takes 
its rise from the centrosome, and is fixed by its attached border along 
the length of the body of the parasite and terminates at the front end, 
while the outer free margin of the membrane is thickened into a rod¬ 
like border which is continued past the front end into the long free 


7 


flagellum. Thus the thick border of the undulating membrane is con¬ 
tinuous behind with the centrosome and in front with the flagellum. 
The web of the membrane is difficult to stain and is seen only faintly 
in stained preparations. In a fresh preparation the body, the undu¬ 
lating membrane, and the flagellum appear to consist of a homogeneous, 
strongly retractile protoplasmic substance possessing marked con¬ 
tractility. 

A disputed point is the biological relation of centrosome to nucleus. 
Rabinowitsch and Kempner, after a study of numerous stained and 
unstained preparations, especially of the developmental forms of the 
trypanosomes, and after an investigation of the views held in regard 
to the nucleus of the flagellata, came to the conclusion that the cen¬ 
trosome and nucleus are an interdependent whole which corresponds 
to the nucleus of the other flagellata. They maintain that in the 
early stage of development of the parasite the centrosome and nucleus 
represent a whole which, as the parasite becomes older, breaks into 
two parts which pass to either end of the adult parasite. They con¬ 
sider the trypanosome’s nucleus as made up of two parts more or less 
separated; the small oval spot situated in the hind end of the try¬ 
panosome they designate the nucleolus, while the larger structure 
found in the front end they call the chromatin heap. 

Wasielewski and Senn (5) think that the centrosome is in no way 
whatever connected with the nucleus. They consider the trypanosome 
as made up of two parts, the “plasma” and the “periplast.” The 
plasma is the body of the parasite containing the nucleus. The peri¬ 
plast is the outer covering of the trypanosome and embraces the cen¬ 
trosome, undulating membrane, flagellum, and an outer coat invest¬ 
ing the body of the parasite. According to this view the centrosome 
is intimately connected with the undulating membrane. It is the 
root from which the thickened free border of the undulating mem¬ 
brane springs and has nothing to do with a nucleolus, as Rabinowitsch 
and Kempner point out, nor with a micro-nucleus, as held by Plimmer 
and Bradford. In their illustrations the outer coat of the parasite 
takes a different stain from the body plasma. 

Laveran and Mesnil mention that blood kept some time shows try¬ 
panosomes reduced to centrosome and flagellum, and that the con¬ 
tinuity of the centrosome and flagellum can be seen. We have seen 
the same thing in stained preparations of fresh blood. 

CYCLE OF DEVELOPMENT. 

We kept fresh blood in hanging drop at room temperature and at 
37° C. and watched it for hours without seeing any of the parasites 
advance in development beyond the stage which they presented when 
the blood was first drawn. The different stages of development can 
be seen only with the help of animal experiments. It is very doubt¬ 
ful if multiplication of the organisms occurs outside the body. 


8 


Rabinowitsch and Kempner were the first to employ white rats inoc¬ 
ulated intraperitoneally for the study of development, and they were 
able to trace the entire cycle of development by the examination at 
short intervals of the blood of a white rat so infected. By a similar 
examination of a great many rats we were able to get all the forms of 
development. 

Multiplication by transverse division. —The first thing to be observed 
is a change in the outlines of the trypanosome. Its sharp beak becomes 
blunt; the flagellate end is no longer sharp, but rounded; the slender 
body becomes thickened and swollen; both the longitudinal and the 
transverse diameters of the parasite are increased. There is a multi¬ 
plication of nuclei and centrosomes to a number which seldom exceeds 
five each, and they are arranged in a line parallel to the long axis of 
the parent trypanosome. After the division of nuclei and centrosomes, 
the next step is the formation of the new flagella, each of which takes 
its origin in a centrosome and generally emerges from the parent try¬ 
panosome on the side which bears the undulating membrane (PI. Ill, 
fig. 9). These new flagella gradually attain full length. The old fla¬ 
gellum becomes destroyed and disappears. The cleavage of the pro¬ 
toplasm of the parent occurs along lines at right angles to the long 
axis, so that each new segment of protoplasm is equipped with a 
nucleus, a centrosome, and a flagellum. 

The daughter cells are soon free in the blood and are readily dis¬ 
tinguished from the adults by their small size and oval outline. They, 
however, gradually lengthen out into the familiar picture of the adult. 

Multiplication by longitudinal division. —This form of multiplication 
has much in common with the transverse division. There is the same 
change in outline of the parent and the same multiplication of cen¬ 
trosomes and nuclei to a number of from two to six. The arrange¬ 
ment of the latter, instead of being in a line parallel to the long axis 
of the parent, is in a transverse line. The new flagella next appear. 
They arise at the front end of the parent and are arranged closely 
about the old flagellum and correspond in number to the centrosomes. 
(PI. I, fig. 3.) Rabinowitsch and Kempner, in speaking of the origin 
of the new flagella, mention that in the flagellata the formation of the 
new flagella takes place so rapidly that the investigators can not fol¬ 
low the process accurately in all its details. They do not hold to the 
view that flagella multiply by a cleavage in the long axis of the old 
flagellum. It is their opinion that the new flagella arise from the 
body plasma and their principal support for such an origin is that in 
transverse division the new formation of flagella does not occur at 
the end close about the old flagellum, but on the side at a consider¬ 
able distance away from the old flagellum. 

Multiplication by segmentation. —These three forms of division are 
separated only for description. They may all be seen side by side in 
the same preparation and in some instances the distinction between 
them is not altogether clear, especially between the longitudinal and 


9 


transverse modes. Division by segmentation begins with a curving 
of the parasite which continues until the two ends approach each 
other and finally meet, thus giving the parasite a globular form. The 
flagellum becomes lost and the undulating membrane disappears. 
The nuclear multiplication now occurs and proceeds in the same man¬ 
ner as in the longitudinal and transverse division. The nucleus 
becomes swollen, appears coarsely granular, and divides into two 
parts, each of which probably divides and subdivides. 

In stained preparations Rabinowitsch and Kempner saw small 
bodies in the new nuclei. These bodies, which were about the size of 
centrosomes, took the stain deeply and were placed at the periphery 
of the nucleus. Whether centrosomes can arise from nuclei or only 
from other centrosomes is a point of controversy and is mentioned 
under morphology. 

Whatever may be the source of the centrosomes their number 
always keeps pace with the number of nuclei. No matter how many 
are formed there is a formation of a corresponding number of centro¬ 
somes until in the end there are as many centrosomes as nuclei in the 
trypanosome. 

As regards the manner of the arrangement of nuclei and centro¬ 
somes in the globular parent, we find them in two concentric circles. 
The outer circle contains the nuclei and the inner circle the centro¬ 
somes. In the cleavage of the protoplasm there are as many segments 
as number of nuclei. While in transverse and longitudinal division 
there are not more than 6 daughter cells, we find in this form as many 
as 16. Plate III, figure 12, shows 14 segments. 

Cleavage proceeds from the periphery to the center of the mass, so 
that a rosette arises as in the segmentation of malaria. Each segment 
of protoplasm contains a centrosome and a nucleus, and a flagellum 
develops from each. In hanging drop these rosettes are seen to be in 
agitation as soon as they acquire their flagella. Finally the segmen¬ 
tation is complete and the daughter trypanosomes are seen as small 
oval young forms free in the blood. Rabinowitsch and Kempner, in 
speaking of segmentation, refer to the hanging together of the young 
by their sharpened ends and their gradual separation from each other; 
but they maintain that the young parasites may develop into adults 
before the radial arrangement is broken up, as is shown in their figure 
27 (Rabinowitsch and Kempner, 1899, pi. 3, fig. 27.) We have not 
seen them develop into adults while so arranged. The rosettes seem 
to become dissolved while their members are still young. We should 
think that figure 27 represented an autoagglutination of adults rather 
than an advanced stage of a rosette, for the reason that the members 
are evidently adults instead of young forms. 

THE DURATION OF THE INFECTION. 

Rabinowitsch and Kempner found that artificially infected white 
rats harbor these parasites in general from four to six weeks. They 


10 


saw them disappear from the blood in some cases in from one to two 
weeks, sometimes earlier. Two rats retained their parasites three to 
four months. 

Wasielewski and Senn found trypanosomes five and one-half months 
after injection in several instances and no animal lost his parasites 
before six weeks. 

Jurgens (6) found that his animals kept their blood parasites as a 
rule one to two months, seldom later. In two cases numerous para¬ 
sites were found after seven months. Only once did he find the 
infection to last shorter than one month. 

We found the duration of the infection to be seven to fourteen days. 
The long periods of infection of three to six months, or even of six 
weeks, we have not seen. The trypanosomes never returned spon¬ 
taneously to the rats after they had once become free of them. We 
were unable to find any wild rats infected spontaneously, but the 
duration of such an infection is, according to Rabinowitsch and 
Kempner, much longer than an artificial infection of white rats. 
They state that in an infection of the wild rats the parasite, so far as 
they have observed, did not disappear from the blood. 

MOTION OF THE TRYPANOSOME. 

If a thin film of fresh blood is watched beneath a cover slip on a 
slide the parasites are seen to be in striking activity, darting among 
the corpuscles and pushing them aside, but never entering the cor¬ 
puscles or engulfing them. The motion is too violent to be closely 
followed by the eye, so in order to gain an accurate perception of the 
mechanism of the motion the film must be very thin, so that the 
trypanosome will be subjected to the pressure between the cover and 
slide, and consequently his motion becomes very slow. 

We see that the parasite generally moves with the flagellum (anterior 
end) in front, but if he meets an obstruction he withdraws for only a 
short distance in the direction of his blunt (posterior) end, and then 
resumes in his original direction. The movement takes place by 
means of the undulating membrane and the flagellum. When in 
motion the body of the parasite rotates on its longitudinal axis, thus 
moving in a screw-like manner. This causes the undulating mem¬ 
brane to appear as if it were spirally arranged around the organism. 
The waves, starting in the flagellum and traveling along the undulat¬ 
ing membrane, are plainly seen. Motion will persist in a cover glass 
preparation for several hours. 

SYMPTOMS IN RATS. 

Rabinowitsch and Kempner mention loss of appetite, slight loss of 
weight and debility, but say that none died from the infection. They 
regard the disease in white rats as mild compared with the same in 
wild rats. 

Jurgens reports severe sickness and death. 


11 


Many of our rats died. Plain evidences of sickness coupled with 
heavy blood infection were almost sure to give a fatal result. We 
made no pathological study of the internal organs. 

STAINING. 

In the beginning we attempted to stain the parasites with the ordi¬ 
nary aniline dyes. We used eosin, methylene blue, hemalum, carbo- 
fuchsin, and carbo-thionin, but all were very disappointing. We 
then turned to the Romanowsky double stain of methylene bine and 
and eosin, Goldhorn’s polychrome methylene blue and Jenner’s stain, 
all of which give most beautiful effects indeed; perhaps the best is 
Romanowsky’s stain. Some modification of this stain is the one which 
many of the investigators have employed within the last few years for 
trypanosomes. In the hands of the various experimenters this stain 
seems to give somewhat different effects. The two points on which 
they all agree are that the nucleus takes a beautiful rose red and that 
the protoplasm takes a blue color. Rabinowitsch and Kempner state, 
and it is clearly shown in their figures, that in the hind part of the 
adult parasite there is an oval or round spot of a homogeneous struc¬ 
ture which takes the stain uniformly and intensely red. In the front 
end of the trypanosome is a larger body with a net-like structure 
which stains rose red. In their hands the protoplasm of the body and 
the free outer border of the undulating membrane and the flagellum 
stained quite uniformly blue. The web of the membrane remained 
unstained. 

Wasielewski and Senn state that the protoplasm is almost homo¬ 
geneous, stains a light blue, and has a fine granular structure. In it 
are one to three clear, oval areas, generally in front of the centrosome, 
which can be considered as vacuoles. With the Romanowsky stain 
they succeeded in giving a red tint, not only to the centrosome and 
nucleus, but to the free border of the undulating membrane, to the 
web of the undulating membrane, to the flagellum, and to the entire 
border of the body of the parasite. They state, however, that the 
tint of the centrosome, flagellum, undulating membrane, and “peri¬ 
plast ” was a bluish red. 

Laveran and Mesnil picture the centrosome and body of the para¬ 
site as blue, and the nucleus, flagellum, and undulating membrane as 
red. With Romanowsky’s stain we gave a blue color to the body of the 
trypanosome. The nucleus took a rose red, and the centrosome was 
deep red. The flagellum and border of the undulating membrane 
were red, and the web of the undulating membrane was faintly red. 

Wright's modification of Romanowsky's stain .—Wright (7), in 
speaking of Romanowsky’s stain, which came out in 1891 as a differ¬ 
ential stain for the chromatin and cytoplasm of the malarial parasite, 
refers to the difficulties and uncertainties attending the preparation 
of the stain until it became finally modified by Leishman (8), whose 


12 


method Wright has further simplified. Wright, in his directions for 
preparing the stain, says to add 1 per cent methylene blue to a one- 
half per cent solution of sodium bicarbonate and steam the mixture 
in an Arnold steam sterilizer for one hour, which renders the methy¬ 
lene blue polychromatic. When cold he adds eosin until the color 
changes from blue to purple and a metallic scum forms on the sur¬ 
face and a black precipitate appears in suspension. The precipitate 
is collected on a filter, dried, and dissolved in methyl alcohol. 
Wright gives the following summary for using the stain: 

1. Make films of blood, spread thinly, and allow them to dry in the air. 

2. Cover the preparation with the alcoholic solution of the dye for one minute. 

3. Add to the alcoholic solution of the dye on the preparation sufficient water, 
drop by drop, until the mixture becomes semitranslucent and a yellowish metallic 
scum forms on the surface. Allow this mixture to remain on the preparation for 
two or three minutes. 

4. Wash in water, preferably in distilled water, until the film has a yellowish 
or pinkish tint in its thinner or better spread portions. 

5. Dry between filter paper and mount in balsam. 

Goldhorn’s stain (9 ).—Dry the film and fix in pure methyl alcohol 
fifteen seconds, wash in running water, stain in 0.1 per cent aqueous 
solution of eosin for thirty seconds, wash in running water, stain one 
minute in Goldhorn’s polychrome methylene blue, wash in water, dry 
in air, mount in balsam. 

The polychrome methylene blue is made as follows: 

1. Dissolve 2 grams of methylene blue and 4 grams of lithium car¬ 
bonate in 300 c. c. of warm water. 

2. Heat in porcelain dish in a boiling water bath fifteen minutes. 

3. Pour into a glass-stoppered bottle and set aside for several days. 

4. Render only slightly alkaline by adding 4 to 5 per cent acetic acid 
solution. 

With this stain we have obtained beautiful preparations showing 
the chromatin of the ring form of sestivo-autumnal malaria and the 
chromatin of the tertian parasite. We have also well-stained prepa¬ 
rations of blood platelets. It is a good stain for the nuclei of animal 
parasites. It shows the chromatin of the segmenting bodies of 
malaria, the chromatin of the crescents, the eosinpliilic and neutro¬ 
philic granules and nuclei of leucocytes. Mast cells and myelocytes 
are well stained. 

Jenner’s stain .—This stain is not so good for trypanosomes as the 
other two. Equal parts of 1 per cent aqueous solutions of eosin and 
methylene blue are mixed and set aside for twenty-four hours. The 
mixture is filtered, the precipitate is washed with water and dried 
and then dissolved in methyl alcohol. In using this stain no previous 
fixing is necessary. After staining one to three minutes the speci¬ 
men is thoroughly washed until the corpuscles appear pink. Dry in 
the air and mount in balsam. 

The stain can be bought ready for use from dealers, or a powder 


13 


can be gotten from Griibler, which is to be dissolved in methyl 
alcohol. 

W e have gone at some length into these stains, for without some 
one of them the investigator will attain little in ihe way of getting 
instructive preparations of trypanosomes and because their more gen¬ 
eral use may bring out some points still unmentioned in the structure 
of animal parasites generally. Since a thorough understanding of 
the stains is necessary to fine work, the reader is referred to the orig¬ 
inal articles mentioned in the bibliography. 

ACTIVE IMMUNITY. 

With very few exceptions a single infection with trypanosomes ren¬ 
ders the rats free from parasites thereafter. Rabinowitsch and 
Kempner had no second infections following the injection of heavily 
infected trypanosome blood into the peritoneal cavity of rats, which, 
after artificial inoculation, had become spontaneously free of para¬ 
sites. Laveran and Mesnil, in their series of thirty, found two suscep¬ 
tible to a second infection. One of our rats infected by feeding and 
another infected by intrastomachal injection proved susceptible to a 
second infection by intraperitoneal inoculation in two and five months, 
respectively, after they had become free of parasites. Their second 
infections lasted only three days. 

PASSIVE IMMUNITY. 

From our knowledge of the antitoxins of the bacterial diseases, we 
are led naturally to the investigation of the protective property of the 
serum of immunized rats, and it is found that there is produced a 
specific immune serum. The serum of rats which have been immu¬ 
nized by one or more inoculations of trypanosome blood does give pro¬ 
tection to other rats within certain limits. Laveran and Mesnil found 
that their most active serum came from a rat which had been given 13 
inoculations. 

If we add in vitro 1 c. c. of immune serum to 1 c. c. of trypanosome 
blood and inject the mixture into a fresh rat, no infection will follow. 
We also separated the two injections in time to see whether immune 
serum would prevent infection if injected before the trypanosome 
blood; likewise, whether the immune serum would prevent infection 
if it were injected after the injection of infected blood. Our results 
were somewhat variable, but in general they corresponded to the limits 
of time which have been set by Rabinowitsch and Kempner and con¬ 
firmed by Laveran and Mesnil, namely, that 1 c. c. of immune serum 
injected into a fresh rat twenty-four hours before or twenty-four hours 
after the injection of trypanosomes will prevent infection. They 
found that emulsions of the spleen, bone marrow, liver, or brain con¬ 
ferred no passive immunity. 

The limits of the preventive and curative power of the immune 


14 


serum, although quite narrow, are, however, as wide as we would 
expect. 

Five pregnant rats were inoculated, with a view to finding whether 
their young would acquire an immunity by placental transmission. 
Although the mothers bore a very heavy infection, we could never 
demonstrate any parasites in the fetuses nor did the young show any 
evidences of an increased resistance to subsequent infection. Laveran 
and Mesnil mention one immune rat which had two litters; the first 
litter was immune, the second susceptible. In this connection we 
may mention that no case of placental transmission of malaria has 
been reported in which the possibility of postnatal infection has been 
excluded. 

SUSCEPTIBILITY OF ANIMALS TO TRYPANOSOMA LEWISI. 

The wild rat and the sewer rat are the only animals in which there 
is a spontaneous conveyance to each other. The white and spotted 
rats are susceptible by inoculation only. Young rats are more sus¬ 
ceptible than old ones. Rabinowitsch and Kempner report the fail¬ 
ure to infect pregnant rats. We inoculated five pregnant females, 
and all bore heavy infections. No one has found that the white 
rats harbored trypanosomes spontaneously in their blood. No inves¬ 
tigators have succeeded in infecting other animals with rat trypano¬ 
somes except Laveran and Mesnil, who infected guinea pigs. We 
tried in vain to infect guinea pigs, rabbits, white mice, cats, a dog, 
a goat, and a horse by intraperitoneal inoculation. In one guinea 
pig we found six parasites in the blood twenty-four hours after 
inoculation, but subsequent examinations of the blood showed an 
absence of all parasites, so that this can not be considered a case 
of infection. Laveran and Mesnil, after intraperitoneal injection of 
a guinea pig with 1 c. c. of blood rich in trypanosomes, found multi¬ 
plication forms in the peritoneum two to five days after injection. 
They had numerous failures in bringing about a blood infection, but 
some guinea pigs showed parasites in the blood in the proportions of 
1: 20 and 1: 50 of the red blood corpuscles. The infection was of short 
duration. 

When attempting to infect the various animals other than rats we 
injected large amounts of heavily infected rat blood. In some cases 
we injected a rat’s entire blood. White rats and wild rats are so 
susceptible that only one to three drops of infected blood, mixed 
with a little sterile salt solution or bouillon and injected intraperito- 
neally, will cause a marked infection. 

AUTO-AGGLUTINATION. 

We have brought forward the use of this term to signify the agglu¬ 
tination of a rat’s own trypanosomes while still circulating in his own 
blood. If daily examinations are made of the blood of an infected rat, 


15 


the parasites will be seen to show agglutination during the period of their 
decline in numbers and disappearance from the blood. When auto- 
agglutination is well advanced we see very few parasites occurring 
singly. They are collected into masses; but these masses in turn 
show a tendency to collect close together, which must necessarily leave 
certain drops of blood almost free from all parasites while other drops 
will show typical fields of auto-agglutination. On this account a 
single hanging drop or a single stained preparation taken from a rat 
is not sufficient to give a correct idea of the condition of the parasites 
in the blood. As many as eight to ten slides may be made in which 
will be found only a few scattered parasites, and the next slide will 
show pictures such as are seen in Plate II, figure 8, and Plate IV, 
figures 13 and 14. Figure 13 represents a single focus of agglutina¬ 
tion, and in figure 14 there are three such foci near to each other. 

A close examination of these agglutinations will show that they 
have nothing to do with the rosettes of multiplication. They are 
found in the blood after the period of multiplication has passed, and 
they are made up of adult parasites instead of young forms. We con¬ 
sider these agglutinations as an evidence of the presence of agglutinin 
in the rat’s blood and as an omen of an impending rapid disappear¬ 
ance of the parasites from the blood. We have in the laboratory in 
several instances seen the agglutination of the parasites in a rat’s 
blood for a few successive days before a sudden disappearance of all 
parasites over night. We conclude from this observation that agglu¬ 
tination is a step toward dissolution, and that it foretells the disap¬ 
pearance of the trypanosomes from the blood. 

From a daily study of the blood of numerous cases in which agglu¬ 
tinations of parasites were present we were able to prophesy that 
active immunity was near at hand. 

It is not every case of infection which shows auto-agglutination. 
We would explain its absence on the ground of an insufficient gener¬ 
ation of agglutinin. As will be referred to later, there is a difference 
in the agglutinating power of the immune sera of different rats. As 
soon as a rat loses his parasites he is considered an immune and his 
serum is admitted to have agglutinating power on fresh parasites to 
which it may be added. The agglutinating power of the immune 
serum is, moreover, attributed to the agglutinin which it contains. 
Now, we think that the production of this agglutinin is a gradual 
process which is begun before a rat loses his parasites, and we think 
that just previous to the disappearance of all parasites from a rat’s 
blood there is a short period during which he may generate sufficient 
agglutinin to agglutinate the parasites circulating in his own blood. 
We would offer as a possible explanation of our own cases that per¬ 
haps our trypanosomes were more virulent than those used by other 
observers. This view seems to be supported by the shorter time 
which elapsed between inoculation and heavy blood infection, the 


16 


more rapid disappearance of the parasites from the blood, and the 
greater number of deaths among the white rats. 

The agglutinated parasites have a most orderly arrangement in the 
shape of a rosette, with their posterior ends close around a central 
point and their flagella at the periphery. Each parasite has its own 
fine undulating motion. 

AGGLUTINATION. 

1. By immune serum .—Immunity and agglutinating power go hand 
in hand. As in late years agglutination has been proven for so many 
kinds of bacteria by their specific sera, so trypanosomes are found to 
respond in a somewhat similar way to immune serum. 

Laveran and Mesnil probably made the most complete study of 
agglutination. The trypanosome blood which they used was sub¬ 
jected to defibrination, which of course left the corpuscles and the 
trypanosomes in the serum. We have found that the substitution of 
clotting for defibrination will show the agglutination to much better 
advantage in the hanging drop, since there will be no corpuscles in 
the field to obscure agglutination. The immune blood and the 
trypanosome-bearing blood may be drawn up into separate fine capil¬ 
laries and allowed to coagulate. The clot is then drawn out at one 
end of the tube, leaving the clear immune serum behind in the one 
case and the clear serum containing trypanosomes in the other case. 
Now the dilutions can be made just as in the Widal reaction. We 
have, however, taken another precaution which Laveran and Mesnil 
did not observe. We removed all agglutinin from the trypanosome 
serum before starting the tests. We were led to this by the study of 
au to-agglutination. 

If we should select for our agglutination tests blood in which auto¬ 
agglutination was already noticeable, we would immediately fall into 
the error of getting agglutination with ever so great a dilution of the 
immune serum, because the trypanosomes were agglutinated before 
we began. Again, if we selected blood in which there was no auto¬ 
agglutination, but in which there was considerable agglutinin, but 
still not enough to produce an auto-agglutination, we would still be 
in error if we attempted to determine the agglutinating power of an 
immune serum by testing it on trypanosome-bearing serum which was 
just on the verge of auto-agglutination. We must therefore take 
into consideration one element which does not enter an agglutination 
test on bacteria. The bacterial pure culture has a fixed, uniform 
composition, and until we are able to grow trypanosomes in pure cul¬ 
ture on artificial media we will have to consider the element of agglu¬ 
tinin in the serum which contains the parasites. 

Our plan was to draw the trypanosome blood from the rat, allow it 
to coagulate, draw off the serum containing the parasites, and dilute 
it with plain distilled water and filter it through a porcelain filter 


17 


under the influence of a vacuum. After four or five washings with 
large amounts of water, we let it filter until there remained behind a 
volume of fluid which equaled the original amount of serum. In this 
residue were the trypanosomes free from agglutinin. Dilutions were 
then made of the immune serum, and it was tested on the washed 
parasites. Some immune sera will not agglutinate in a dilution 
greater than 1:1. An agglutinating power of 1:5 or 1:10 is common. 
One of our rats showed typical agglutination in a dilution of 1:200. 

It is interesting to watch in hanging drop an agglutination by a 
weak serum. At first two or three parasites are seen, joined by their 
posterior ends. Others come up toward the center of agglutination, 
recede for some distance, and later join the others. Some disengage 
themselves from the rosette and then rejoin it, until finally a well- 
arranged rosette is formed. Two small rosettes will gradually 
approach each other and then unite to form one mass, which in turn 
is joined by others of smaller or larger size. 

A remarkable fact is that the agglutinated parasites do not lose 
their motility. There is not the diminution of motility before agglu¬ 
tination that is seen in a typhoid reaction, and while agglutinated 
each parasite retains a regular vibration. In agglutination with a 
strong serum the process takes place rapidly. The parasites rush 
together in great numbers, and the masses may be of macroscopic 
size. If a strong specific serum is used, the agglutinated masses are 
very compact and the individual parasites are tightly drawn together, 
so that there is little motion. In a general way the parasites are all 
pointed toward a center, but still they overlap and cross each other 
very much. With a weak serum the parasites are held together in a 
loose manner, permitting of more individual movement to each organ¬ 
ism and more orderly arrangement, and there are fewer parasites to 
each rosette. 

We often found agglutination almost complete, in which case very 
few parasites were to be seen free in the field. It may be only par¬ 
tial. The parasites may remain agglutinated until their death, or, if 
the serum is weak, a disagglutination may follow. Specific sera 
exposed to 55° C. for thirty minutes did not lose the agglutinating 
power, but a temperature of 65° C. maintained for half an hour 
destroyed its activity. 

Laveran and Mesnil found that trypanosomes killed by chloroform 
or formalin were agglutinated by the same sera which agglutinate the 
living, but the parasites have no orderly arrangement in the mass. 
It is a remarkable fact that Rabinowitsch and Kempner found that 
“the trypanosome serum shows in no way whatever the property of 
agglutination. ” 

2. By normal sera .—The action of normal sera in dilution of 1:1 
was tested on the trypanosomes. The cat and horse sera were 
strongly agglutinating. The goat and rabbit sera were feebly agglu- 


20564—No. 11—03-2 



18 


tinating. The sera of the white rat, white mouse, and guinea pig 
were negative. 

Laveran and Mesnil state that the normal sera of the chicken and 
horse caused complete agglutination. The sera of the sheep, dog, 
and rabbit produced a partial reaction, while the pigeon, frog, guinea 
pig, white and spotted rats, and sewer rats caused none whatever. 
They also state that, although the blood of chicken and horse have 
agglutinating power, they do not protect against infection. 

3. In the ice chest .—If trypanosome blood is drawn from the rat 
under aseptic conditions and sealed in pipettes and placed in the ice 
box, the parasites will usually join into beautiful rosettes after 
twenty-four hours. We found them agglutinated in blood which had 
been eighty-three days in the ice chest. 

TRANSMISSION OF THE DISEASE. 

1. By intraperitoneal inoculation ,—Although white and spotted rats 
have never yet been found to harbor trypanosomes spontaneous^ in 
their blood, we find in them a very susceptible host for experimental 
inoculations, and it was with them that most of our work was done. 
Comparative studies show that a heavy blood infection is obtained by 
intraperitoneal injection sooner than by any other form of inocula¬ 
tion. The blood to be injected is mixed with 1 c. c. of saline solution 
or bouillon and injected with a hypodermic syringe. The period 
which elapses between the intraperitoneal inoculation and the first 
appearance of the parasites in the blood is variable, depending upon 
the amount of trypanosomes injected and the stage of develpment of 
the injected parasites. 

Rabinowitsch and Kempner place the time at three to seven days, 
although they observed parasites in a few instances within the first 
day. 

Laveran and Mesnil give three to seven days as the average time 
before the blood infection. They found a few in the circulation after 
five or six hours. 

Jourgens found that the first presence of parasites in the tail blood 
occurred three or. four days after inoculation, seldom later. After 
inoculation with 1 c. c. of blood he found them in tail blood in several 
instances after twenty minutes. 

The parasites appeared in the tail blood in our cases usually on the 
second day. This was chiefly because we injected as a rule larger 
doses and used heavily infected blood in which were many develop¬ 
mental forms. 

There has been considerable discussion as to where the principal 
seat of multiplication takes place in intraperitoneal inoculation. 
Rabinowitsch and Kempner regard the peritoneal fluid as a better 
nutritive medium for the development of the parasites than the blood 
and think that the chief seat of development and multiplication is in 
the peritoneum. In one to five days after intraperitoneal injection 


19 


of trypanosome blood they found in the peritoneum several engaged 
in development. They think that as soon as development is perfected 
in the peritoneum the parasite disappears from the peritoneal fluid 
into the blood. They give some weight to the fact that in one case of 
intravenous injection of parasites multiplication did not occur until 
the fifth day. Laveran and Mesnil state that there is less multiplica¬ 
tion in the blood than in the peritoneum. 

Our observations speak for the blood as the principal seat of devel¬ 
opment. On examination of the peritoneal fluid at varying times 
after injection we did not find division forms, nor did we find the 
first existence of parasites in the tail blood accompanied by division 
forms. The most reasonable explanation seems to be that if a large 
amount (0.75 c. c.) of heavily infected blood be injected into the 
peritoneal cavity, the small young forms and the long slender adults 
pass at once by the lymphatics into the general blood stream in suffi¬ 
cient numbers to be detected in cover-slip preparations within twenty- 
four hours or even within twenty-minutes, but the rosettes and other 
large division forms which are injected into the peritoneal cavity are 
prevented by their size from passage through the lymph channels 
and remain behind in the peritoneal cavity until their division is 
complete, when the young then pass through the lymph passages into 
the blood, leaving the peritoneum permanently free from parasites. 

Daily examination of the blood for one to two days after the first 
appearance of parasites in the tail blood shows at most only a very 
gradual increase in the number of parasites present, but suddenly 
there comes an enormous swarming of the blood with parasites, and 
the presence of rosettes and other division forms indicate that multi¬ 
plication is going on in the blood. The slide from which the micro¬ 
photographs (PI. Ill, figs. 11,12) were made shows at least two dozen 
rosettes and was taken from the rat on the second day after the first 
appearance of parasites in the tail blood. It is not unusual to find 
parasites in the proportion of 1:2 red blood cells. Exceptional 
instances are met with in which the blood corpuscles are outnumbered 
by the trypanosomes. The duration of the period of multiplication 
is often no more than twelve to twenty-four hours. By the fourth 
day after the first entrance of parasites into the blood the height of 
infection has been reached. Rabinowitscli and Kempner found no 
division forms in the blood on the fourth day after the first appear¬ 
ance of parasites in the tail blood. 

2. By subcutaneous inoculation .—Blood infection occurs by this 
form of injection a little later than by intraperitoneal inoculation. 
The shortest time in our cases between inoculation and the appear¬ 
ance of parasites in the blood was three days. Multiplication pro¬ 
ceeded in the blood until it swarmed with myriads of parasites. This 
would seem to be additional evidence to the superiority of the blood 
over the peritoneal fluid as a nutritive medium for the development 
of the parasites, for in cases where the parasites had advantage of 


20 


tlie peritoneal fluid in addition to the blood their number did not 
reach a point beyond the number obtained by subcutateous injection. 

S. By intrastomachal injection .—We have read some discussion 
bearing on the natural mode of infection with trypanosomes in which 
infection followed the eating of a trypanosome rat by a healthy one. It 
was held that this could not be considered a case of intrastomachal in¬ 
fection because the possibility of the entrance of the parasites through 
wounds about the mouth, lips, or teeth had not been excluded. 

We therefore arranged a series of experiments in which we thought 
all likelihood of infection through wounds was removed and that 
infection occurred through the stomach. Twelve white rats were 
chloroformed sufficiently to prevent any struggling. Then a small- 
sLzed catheter well oiled was passed into the stomach without 
encountering any resistance; injection of trypanosomes was made 
through the catheter, and the rats were then placed in separate cages 
and examined daily for parasites in the tail blood. Eleven out of the 
12 developed blood infections fully as heavy as was obtained by any 
other form of inoculation. The time which elapsed before their 
appearance in the blood was as follows: Two in four days, one in five 
days, three in six days, three in seven days, and two in eight days. 

We see that infection is considerably delayed by this method, the 
earliest being in four days and the latest in eight days. 

J/.. Transmission by feeding .—The success of the intrastomachal 
injections naturally lead to a series of experiments to determine 
whether infection would not occur by feeding when all precautions 
were taken to prevent any wounding about the teeth or mouth. 

Seven white rats, apparently free from mouth wounds, were put 
into separate cages to prevent fighting, and they were fed with soft 
food, so that no wounds would result from the gnawing of bones. 
They were each given a single feeding with the entire blood of a 
trypanosome rat. No other part of the infected rats was given to 
them. Trypanosomes appeared in the tail blood in five of the seven 
at periods of time which averaged six days. 

Wild rats were then subjected to similar feeding experiments. In 
the blood of five wild rats we found parasites after three, seven, eight, 
nine, and ten days. Some of the rats had enormous numbers in their 
blood, while others had comparatively few. 

We conclude from these experiments that infection may take place 
through the digestive tract and that the spread of the disease among 
wild rats may be due to feeding upon one another, especially since 
the instinct of fighting and pluck is so well implanted in them and is 
brought into action on slight provocation. We found it necessary to 
separate the wild rats in our stock cage to prevent losses from injuries 
inflicted on the weaker ones by the stronger. 

After beginning the feeding experiments we were surprised to read 
the results obtained by Rabinowitsch and Kempner. They made intra¬ 
stomachal injections through a stomach tube in four rats, but were 


21 


unsuccessful, although they repeatedly introduced into one rat blood 
rich in parasites and nourished one rat chiefly by this material. They 
placed together an infected tame rat and a noninfected wild rat, and 
state that after a hot combat the tame one was killed by the wild rat 
and eaten with much pleasure. Parasites appeared in his blood after 
ten days. They did not regard this as a case of infection necessarily by 
the digestive tract, as the parasites may have entered by the numerous 
bites and wounds, nor do we regard it as such; but we think we have 
in our experiments removed the likelihood of entrance through wounds 
and have established the existence of infection through the digestive 
tract alone. 

5. Transmission by fleas. —Rabinowitsch and Kempner, with a view 
to finding the natural mode of transmission, placed together an infected 
white rat with a healthy white rat. The latter became infected in 
eleven days. They repeated the experiment and found parasites in 
the blood of another rat after fifteen days. A gray rat showed para¬ 
sites in his blood after fifteen days’ confinement with two infected 
animals. The three rats which became infected had many fleas on 
them. Examination of a great number of teased preparations of the 
fleas did not reveal any of the trypanosomes in them. They then 
mashed up fleas collected from infected rats and injected this material 
into the peritoneal cavity of nine white rats. Five became infected. 
Likewise four rats were injected intraperitoneally with mashed-up lice, 
but no infection followed. The next experiment was to determine 
whether the bites of fleas were infective. Twenty fleas were collected 
from infected rats and placed on one healthy white rat, which after three 
weeks time was found to be infected with trypanosomes. They say 
that from this one positive experiment they conclude that fleas can carry 
trypanosomes, and in the absence of proof of another way of conveyance 
they are of the opinion that fleas are the ordinary medium of infection. 

Jourgens states that his experiments were not then complete, but 
that he had kept infected rats and healthy rats together in the same 
cage without infection taking place, although the animals were 
strongly beset with fleas, while a later inoculation of the sound ani¬ 
mals proved them to be susceptible. 

6. Trypanosomes in lice. —Laveran and Mesnil found trypanosomes 
in the stomachs of lice which infested infected rats, but do not report 
a conveyance of the disease by lice bites. 

7. Trypanosomes in Stegomyia fasciata. —We may be pardoned for 
mentioning a subject which is entirely outside of rat trypanosomes— 
but it may have some future bearing on the disease—to state that 
within a year Durham (10) has reported finding trypanosomes in a 
mosquito, thus adding one more to the rapidly growing list of diseases 
through which this little creature threatens the public health. Dur¬ 
ham’s report is inserted. 

A small bat (Phyllostoma) which could not be examined at once was placed in 
a gauze cage, and with it a specimen of Stegomyia fasciata. The next day the bat 


22 


was dead and the mosquito full of fresh blood. This blood contained abundant 
trypanosomes, whose shape is quite different from the usual ones found in rats, 
Nagana, etc. * * * Although flagellates, coccidia-like bodies, etc., often were 
found from time to time in the 80 mosquitoes which were dissected, this was the 
only time that trypanosomes were found. 

THE PARASITES OUTSIDE THE BODY. 

We must confirm the reports of Wasielewski and Senn, and of 
Rabinowitsch and Kempner, that if hanging drops are made of blood 
containing parasites in different stages of division a careful watching 
of the specimen will not witness the completion of the division process, 
whether the specimens be kept at room temperature or in the ice box 
or in the incubator. 

Jurgens found quite the contrary. He made three hanging drops 
of the same blood with the same platinum loop. No. 1 was placed at 
37° C. No. 2 was kept at room temperature. No. 3 was immediately 
dried, fixed, and stained, and examination showed it to contain para¬ 
sites preparing to divide. On the next day Nos. 1 and 2 were dried, 
fixed, and stained. The stained specimen of No. 2 always showed 
the same conditions as No. 3; but in the stained specimen of No. 1 
there were rosettes and division forms, none of which was seen in 
No. 3. Therefore, he concludes that it is possible for certain stages 
of the parasite under certain conditions to increase outside the body. 

It will require further investigation to determine whether the para¬ 
site exists in the flea’s body in the forms known to us as young forms, 
division forms, and adult parasites, or whether there is still another 
form which has not yet been described. The fact of not being able 
to find in infective fleas any form resembling a trypanosome has a 
close parallel in what occurred in one of our rats. A white rat in 
whose blood we saw many parasites in agglutination for several days 
suddenly showed an absence of parasites from the blood. We imme¬ 
diately performed an autopsy and made a careful search of the lungs, 
liver, spleen, kidneys, heart, brain, and bone marrow for parasites, 
but could find none. We were struck by the amount of granular 
debris in the liver and kidneys, but could not see any trypanosomes 
in them. 

An emulsion was made of the kidneys and injected into the perito¬ 
neal cavity of a young rat. The liver was treated in the same man¬ 
ner and injected into two young rats. On the ninth day all three 
rats had parasites in their blood and a few days later developed 
heavy infections. While some unknown form of the parasite may 
have been present, it is of course possible that well-known forms of 
the trypanosomes were in the kidneys and liver and escaped notice. 

Jurgens produced infection with 0.000005 c. c. of blood. If this 
amount of blood be added to 1 or 2 c. c. of salt solution, the dilution 
of the parasites would be so great that they might readily be missed in 
hanging-drop preparations. 

The possibility of the existence of some very minute form of the 


23 


parasite was perhaps excluded by our filtration experiments. Try¬ 
panosome blood was diluted with fifteen parts of physiological salt 
solution and subjected to a porcelain filter under the influence of a 
vacuum. The filtrate was found to be noninfective to rats. 

In blood kept in the ice chest for eighty-one days we found living 
trypanosomes. Their motion was of a trembling, vibrating character. 
Very few crossed the field. Many were arranged in rosettes. In the 
stained preparations we found parasites in which the positions of the 
centrosome and nucleus were reversed, the centrosome being anterior 
to the nucleus. In many of the trypanosomes there was granular 
degeneration, and there was also much free granular debris, which 
probably represented the remains of degenerated parasites. At room 
temperature and at 37° C. the parasites are short-lived. The different 
investigators found them capable of producing an infection of rats 
after being kept in the incubator and at room temperature from four 
to seven days. The parasites when kept at 12° to 16° C. in sterile 
pipettes can maintain their vitality very much longer than under any 
other condition. 

Laveran and Mesnil kept parasites alive in defibrinated blood or in 
equal parts of defibrinated blood and physiological salt solution for 
forty-seven days in the ice box. At the end of this time the blood 
was injected into rats and produced a blood infection in nine days. 
In blood kept under the same conditions for fifty-one days they saw 
no parasites by microscopic examination, but it was infective for,rats. 

Jurgens found only a few parasites in blood kept in the ice chest 
for fifty-three daj T s, but rats injected with it showed parasites in their 
tail blood after seven days. 

Bacteria have a very detrimental effect on the life of the parasites 
outside the body. In hanging drops of trypanosome blood which 
has become infected with bacteria the parasites rapidly die. Blood 
drawn for keeping in the ice box must be kept under sterile condi¬ 
tions. Trypanosomes are killed after an exposure of a few minutes 
at 55° C. Wasielewski and Senn found living trypanosomes in the 
bloody urine of a rat. Rabinowitsch and Kempner were never able 
to find the parasites in the feces or urine. 

THE TRYPANOSOME OF THE HAMSTER. 

This animal harbors a parasite which has been studied by Rabino¬ 
witsch and Kempner. They find that it resembles the rat trypano¬ 
some in that it can hardly be differentiated from it morphologically 
and has the same process of development. On the other hand, they 
could not convey it to rats, and some of the rats which were refractor 
to hamster trypanosomes later proved susceptible to rat trypanosomes. 

These facts, taken together with the fact that they could not convey 
rat trypanosomes to the hamster, led them to conclude that the ham¬ 
ster trypanosome and the rat parasite represent two different physio¬ 
logical varieties, which morphologically are almost inseparable. 


24 


TRYPANOSOMIASIS IN MAN. 

Trypanosomiasis as a disease of man has not yet become acclimatized 
in the United States. At least no cases have been reported in this 
country. There have appeared in the foreign journals within the 
past year, however, eleven authentic instances of infection with 
trypanosomes. 

A case of trypanosomiasis in a European , by Dutton (11) and Forde 
(12).—The patient was a European, 42 years of age, master of a gov¬ 
ernment steamer on the Gambia River, in West Africa. 

On May 10, 1901, the patient was admitted into the hospital at 
Bathurst, West Africa, suffering from what was regarded as malarial 
fever. Examination of his blood did not show malarial parasites, but 
there were seen extremely active bodies which were regarded as filaria. 
Three weeks later the patient was invalided to Liverpool, but returned 
in December, 1901, to Bathurst, where Dr. Dutton, of the Liverpool 
School of Tropical Medicine, examined the patient’s blood and found 
the same parasite which Forde had probably seen seven months pre¬ 
viously and which he at once recognized as a trypanosome. 

The symptoms were an irregularly intermittent fever, a very marked 
erythema multiforme of the trunk and limbs, an oedematous condition 
of the face beneath the eyes and of the ankles, an acceleration of 
respiration and pulse rates, debility and loss of flesh, and enlarged 
spleen. The symptoms persisted throughout the eight months during 
which he was under observation and showed no reaction to treatment 
further than a slight abeyance under Fowler’s solution. 

Dutton found, while making examinations of fresh blood during the 
month of December, 1901, an average of one and one-half trypano¬ 
somes to each cover slip preparation. One preparation showed as 
many as 15 parasites. This case continued in its chronic course until 
the last week of life, during which week the disease assumed an 
acute type and the patient died January 1, 1903. 

Dutton suggests the name Trypanosoma gambiense in case that 
further study shows it to be a new species. 

A second case of trypanosomiasis in a European , by Manson (13).— 
The patient was the wife of a missionary on the Upper Kongo, where 
she had lived for a year. On account of sickness she returned to 
London. 

Dr. Manson, of the London School of Tropical Medicine, recogniz¬ 
ing the same group of symptoms which the patient of Dutton and 
Forde presented, made systematic, careful examinations of her blood 
daily for two weeks. During the two weeks no trypanosomes were 
found, but at the end of this time the parasites were readily seen in 
the peripheral circulation. 

Trypanosomes in the blood of a West Africa native , by Dutton 
(14).—Three trypanosomes were found in a single smear from the 


25 


blood of a child 3 years of age, a native of Gambia. The child showed 
no symptoms of disease. 

Eight additional cases of human trypanosomiasis .—In the British 
Medical Journal for February 7,1903, there is a “preliminary account 
of the investigations of the Liverpool expedition to Senegambia,” by 
Dutton, Annett, and Todd. They found trypanosomes in the blood 
of a white trader who had been twenty years in Gambia. The highest 
number found in a fresh preparation was seven. The patient had 
lost 45 pounds within the past year, complained of weakness and 
breathlessness, and had slight fever at times. The spleen was 
enlarged and there was pitting of the ankles. Four other cases of 
infection of natives were found. 

Three more cases of human trypanosomiasis are reported in the 
British Medical Journal for March 28, 1903, by Dr. Patrick Manson. 
They came from the European community on the Kongo, as did also 
Dr. Manson’s first case. A blotchy erythema and attacks of fever 
characterized these cases. 

The finding of trypanosomes in man, associated with a well-defined 
group of signs and symptoms, is no small contribution to the disen¬ 
tanglement of the diseases of the Tropics. These cases will lead to 
the recognition of others, perhaps, in the tropical parts of our own 
continent or of Asia. The disease has been found in West Africa, 
and with this new fact in parasitology before us its geographical dis¬ 
tribution may be found to have a much wider range. On account of 
the interest which surra claims in the Philippines and on account of 
the recognition within the past year of trypanosomes in man, and 
since the process of development and conveyance of these blood para¬ 
sites have heretofore been little investigated in this country, we have 
undertaken this study. 

We wish to call attention to the autoagglutination, transmission by 
feeding, transmission by intrastomachal injection, and to the staining. 

I am glad to thank the director of the laboratory, Dr. M. J. Rose- 
nau, for his interest in outlining the work. 

I am indebted to Dr. John F. Anderson, the assistant director, for 
valuable suggestions, especially in the staining of the parasites. 

I also desire to express my thanks to Dr. H. B. Parker for making 
the microphotographs. 

REFERENCES. 

1. Salmon, D. E., and Stiles, Ch. Wardell: Emergency report on surra. Bureau 

of Animal Industry, Bulletin No. 42. Washington, 1902. 

(This bulletin contains a complete bibliography of surra and allied trypanosomatic 
diseases by Albert Hassall.) 

2. Crookshank: Flagellated protozoa in the blood of diseased and apparently 

healthy animals. Journ. Royal Microscop. Soc., November, 1886. 

3. Rabinowitsch, Lydia, and Kempner, Walter: Beitrag zur Kenntniss der Blut- 

parasiten, Speciele der Rattentrypanosomen. Zeitschrift fur Hygiene, Vol. 

XXX, 1899. 


26 


4. Laveran and Mesnil: Recherches morphologiques et experimentales sur le 

Trypanosome des rats. Ann. de l’lnst. Pasteur, vol. 15, 1901. 

5. Wasielewski and Senn: Beit rage zur Kenntniss der Flagellaten des Ratten- 

blutes. Zeitschrift fur Hygiene, vol. 33, 1900, p. 444. 

6. Jurgens: Beitrag zur Biologie der Rattentrypanosomen. Archiv fur Hygiene, 

band 42, heft 3, 1902. 

7. Wright, James H.: A rapid method for the differential staining of blood 

films and malarial parasites. Journ. of Med. Research, Boston, vol. 7,1902. 

8. Leishman: Brit. Med. Journ., Sept. 21, 1901. 

9. Goldhom, L. B.: A new and rapid method for staining the chromatin of the 

malarial parasite; a new blood stain. The Hew York Univ. Bull, of Med. 
Sci., vol. 1, No. 2, April, 1901. 

10. Durham, H. E.: Thompson-Yates Laboratories Report, vol. 4, part 2, 1902, p. 

563. 

11. Dutton, J. Everett: Preliminary note upon a trypanosome occurring in the 

blood of man. Thompson-Yates Laboratories Report, vol. 4, part 2, May, 
1902. Liverpool. 

12. Forde, R. M.: Some clinical notes on a European patient in whose blood a 

trypanosome was observed. Journ. Trop. Med., vol. 5, No. 17, Sept. 1,1902. 
London. 

13. Manson: Journ. Trop. Med., vol. 5, No. 21, p. 330. London. 

14. Dutton, J. Everett: Thompson-Yates Laboratories Report, vol. 4, part 2, May, 

1902, footnote p. 467. 
















PLATE I. 


Drawn with Abbe drawing camera. Stained according to Romanowsky method. 

X 1,000. 

Figs. 1 and 2. Adnlt trypanosomes. 

Fig. 3. Parasite undergoing longitudinal division. 

Fig. 4. Transverse division. 


Plate I 



FIG.l. 


FIG. 2. 





\J ' I 


FIG.3. 


FIG. 4. 


























































■* 




















* 









































PLATE II. 


Fig. 5. Beginning multiplication. 

Fig. 6. Same, later stage. The mother parasite is still visible. 
Fig. 7. A multiplication rosette. 

Fig. 8. Auto-agglutination. 


30 


Plate II 






FIG. 6. 



FIG.8. 




























PLATE III. 


Microphotographs. Stained with Goldhom’s stain. 

Fig. 9. Transverse division. The two new flagella coming off from the side which 
bears the undulating membrane. 

Fig. 10. Advanced stage of division. The parent parasite is curved around a red- 
blood corpuscle. 

Figs. 11 and 12. Multiplication by segmentation. The rosettes are composed of 9 
and 14 daughter parasites. 


32 















































i 














































































































20564—No. 11—03-3 



PLATE IV. 

Auto-agglutination. 

Fig. 13. Shows a single focus of agglutination. 
Fig. 14. Shows three such foci. 

34 


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